Vaccines which induce a cell-mediated immune response are emerging as important strategies in combating parasites, autoimmune disorders, allergic diseases and cancers. Conventional vaccination strategies generally involve administration of either “live” or “dead” vaccines. Ertl et al. (1996) J. Immunol. 156:3579–3582. The so-called live vaccines include attenuated microbes and recombinant molecules based on a living vector. The dead vaccines include those based on killed whole pathogens, and subunit vaccines, e.g., soluble pathogen subunits or protein subunits. Live vaccines are generally successful in providing an effective immune response in immunized subjects; however, such vaccines can be dangerous in immunocompromised or pregnant subjects, can revert to pathogenic organisms, or can be contaminated with other pathogens. Hassett et al. (1996) Trends in Microbiol. 8:307–312. Dead vaccines avoid the safety problems associated with live vaccines; however such vaccines often fail to provide an appropriate and/or effective immune response in immunized subjects.
More recently, direct injection of plasmid DNA by intramuscular (Wolff et al. (1990) Science 247:1465:1468) or intradermal injection with a needle and syringe (Raz et al. (1994) PNAS USA 91:9519–9523) has been described. Another approach referred to as ballistic or particle-mediated DNA delivery employs a needless particle delivery device to administer DNA-coated microscopic gold beads directly into the cells of the epidermis. (Yang et al. (1990) PNAS USA 87:9568–9572). Thus, a number of delivery techniques can be used to deliver nucleic acids for immunizations, including particle-mediated techniques which deliver nucleic acid-coated microparticles into target tissue (see, e.g., co-owned U.S. Pat. No. 5,865,796, issued Feb. 2, 1999). Particle-mediated nucleic acid immunization techniques have been shown to elicit both humoral and cytotoxic T lymphocyte immune responses following epidermal delivery of nanogram quantities of DNA. Pertmer et al. (1995) Vaccine 13:1427–1430. Such particle-mediated delivery techniques have been compared to other types of nucleic acid inoculation, and found markedly superior. Fynan et al. (1995) Int. J. Immunopharmacology 17:79–83, Fynan et al. (1993) Proc. Natl. Acad. Sci. USA 90:11478–11482, and Raz et al. (1994) Proc. Natl. Acad. Sci. USA 91:9519–9523.
A novel transdermal drug delivery system that entails the use of a needleless syringe to deliver solid drug-containing particles in controlled doses into and through intact skin has also been described. In particular, commonly owned U.S. Pat. No. 5,630,796 to Bellhouse et al., describes a particle delivery device (e.g., a needleless syringe) that delivers pharmaceutical particles entrained in a supersonic gas flow. The particle delivery device is used for transdermal delivery of powdered drug compounds and compositions, for delivery of genetic material into living cells (e.g., gene therapy) and for the delivery of biopharmaceuticals to skin, muscle, blood or lymph. The device can also be used in conjunction with surgery to deliver drugs and biologics to organ surfaces, solid tumors and/or to surgical cavities (e.g., tumor beds or cavities after tumor resection). Pharmaceutical agents that can be suitably prepared in a substantially solid, particulate form can be safely and easily delivered using such a device.
One particular particle delivery device generally comprises an elongate tubular nozzle having a rupturable membrane initially closing the passage through the nozzle and arranged substantially adjacent to the upstream end of the nozzle. Particles of a therapeutic agent to be delivered are disposed adjacent to the rupturable membrane and are delivered using an energizing means which applies a gaseous pressure to the upstream side of the membrane sufficient to burst the membrane and produce a supersonic gas flow (containing the pharmaceutical particles) through the nozzle for delivery from the downstream end thereof. The particles can thus be delivered from the needleless syringe at delivery velocities of between Mach 1 and Mach 8 which are readily obtainable upon the bursting of the rupturable membrane.
Another particle delivery device configuration generally includes the same elements as described above, except that instead of having the pharmaceutical particles entrained within a supersonic gas flow, the downstream end of the nozzle is provided with a bistable diaphragm which is moveable between a resting “inverted” position (in which the diaphragm presents a concavity on the downstream face to contain the pharmaceutical particles) and an active “everted” position (in which the diaphragm is outwardly convex on the downstream face as a result of a supersonic shockwave having been applied to the upstream face of the diaphragm). In this manner, the pharmaceutical particles contained within the concavity of the diaphragm are expelled at a high initial velocity from the device for transdermal delivery thereof to a targeted skin or mucosal surface.
Transdermal delivery using the above-described device configurations is generally carried out with particles having an approximate size that generally ranges between 0.1 and 250 μm. Particles larger than about 250 μm can also be delivered from the device, with the upper limitation being the point at which the size of the particles would cause untoward damage to the skin cells. The actual distance which the delivered particles will penetrate depends upon particle size (e.g., the nominal particle diameter assuming a roughly spherical particle geometry), particle density, the initial velocity at which the particle impacts the skin surface, and the density and kinematic viscosity of the skin. Target particle densities for use in needleless particle injection generally range between about 0.1 and 25 g/cm3, and injection velocities generally range between about 150 and 3,000 m/sec.
The level of effective protection achieved with DNA-vaccines is similar to that elicited by traditional protein subunit vaccines and killed or attenuated viral vaccines; but is traditionally less than that observed in convalescent animals following recovery from a natural infection. Manickan et al. (1997) Critical Review Immunol. 17:139–154. Herpes infections are extremely prevalent and are caused by two viruses, herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2). HSV-1 is usually acquired in childhood and is the predominant cause of oral infections. HSV-2 infections are usually associated with sexually transmitted genital infections. However, up to 25% of genital herpes is caused by HSV-1. Natural infection appears to impart cross-protection between the two HSV strains, in that, individuals infected with one strain (e.g., HSV-1) have a low incidence of infection with the other strain (i.e., HSV-2) despite exposure. Mertz et al. (1992) Ann. Intern. Med. 116:197–202. The reasons for these observations have not been fully elucidated. Although near complete protection against infection with herpes simplex virus (HSV) can be achieved in mice and/or guinea pigs by vaccination with killed or modified virus, the degree of protection in convalescent animals sets the target for improvement of vaccine performance.
HSV is a double-stranded DNA virus having a genome of about 150–160 kbp. HSV-1 and HSV-2 genomes are colinear and share greater than 50% homology over the entire genome. For some genes, the amino acid identity between the two virus types is as much as 80 to 90%. As a result of this similarity, many HSV-specific antibodies are cross-reactive for both virus types.
The viral genome is packaged within an icosahedral nucleocapsid which is enveloped in a membrane. The membrane (or envelope) includes at least 10 virus-encoded glycoproteins, the most abundant of which are gB, gC, gD, and gE. The viral glycoproteins are involved in the processes of virus attachment to cellular receptors and in fusion of the viral and host cell membranes to permit virus entry into the cell. The glycoproteins are located on the surface of the virion. Consequently, they are targets of neutralizing antibody and antibody dependent cell cytotoxicity (ADCC). Within a virus type, there is a limited (1 to 2%) strain-to-strain sequence variability of the glycoprotein genes. The viral genome also encodes over 70 other proteins, including virion proteins, such as VP16 and VP22 which are associated with the virion tegument, located between the capsid and the envelope. In addition, a group of approximately five ICPs are encoded by the virus. (See, e.g., WO/9516779 regarding ICP4-containing vaccines). These early proteins are synthesized early in the viral replication cycle, in contrast to the envelope glycoproteins which are only made late in the life cycle of the virus.
For a review of the molecular structure and organization of HSV, see, for example, Roizman and Sears (1996) “Herpes simplex viruses and their replication” in Fields Virology, 3rd ed., Fields et al. eds., Lippincott-Raven Publishers, Philadelphia, Pa.
One approach to HSV vaccine development has been the use of isolated glycoproteins which have been shown to be both protective and therapeutic. See, e.g., Burke et al., Virology (1991) 181:793–797; Burke et al., Rev. Infect. Dis. (1991) 13(Suppl 11):S906–S911; Straus et al., Lancet (1994) 343:1460–1463; Ho et al., J. Virol. (1989) 63:2951–2958; Stanberry et al., J. Infect. Dis. (1988) 157:156–163; and Stanberry et al., (1987) J. Infect. Dis. 155:914–920; Stanberry, L. R. “Subunit Viral Vaccines: prophylactic and therapeutic use.” In: Aurelian L (ed.) Herpesviruses, the Immune Systems and Aids. Kluwer, Boston, pp. 309–341. However, clinical trials have failed to demonstrate significant protective immunity in humans vaccinated with a subunit vaccine consisting of the gB and gD glycoproteins.
One method of identifying sequences encoding immunogenic epitopes involves the use of expression libraries, and is known as expression library immunization (ELI). International Publication WO 96/31613 reports that introducing cloned expression libraries from cDNA or fragmented genomic DNA into a subject induces an immune response which can be quantified to select libraries including sequences with immunogenic epitopes. Selected pools are then identified and further characterized. The genomic fragments used are relatively small, between about 10 and 100 base pairs in length. Moreover, in the ELI method, genomic fragments are spliced into a vector having an exogenous (e.g., heterologous) promoter. The genomic clones of ELI remain unidentified sequences and, accordingly, any antigenic sequences must be extensively purified and characterized before a specific vaccine can be developed.
Despite these reports, there remains a need for methods of eliciting an immune response that more closely mimics an animal's natural response to antigens. The present invention provides a solution to this and other problems.